Removal of undesirable genes using yeast backcrossing

Area of Science:

• Food Science
• Microbiology
• Yeast Genetics

Background:

• 4-Vinylguaiacol (4-VG) contributes phenolic off-flavors in brewed liquors, including Japanese sake.
• In sake brewing, 4-VG is produced by the decarboxylation of ferulic acid, catalyzed by yeast enzymes encoded by PAD1 and FDC1 genes.
• While commercial sake yeasts often have non-functional FDC1 alleles, wild yeasts used for specialty brews may retain functional FDC1, leading to higher 4-VG.

Purpose of the Study:

• To develop a breeding strategy for eliminating undesirable wild-type FDC1 alleles from sake yeast strains.
• To reduce 4-vinylguaiacol production in sake by creating yeast strains with homozygous non-functional FDC1 genes.
• To demonstrate a method for targeted gene allele exclusion in yeast for customized strain development.

Main Methods:

• Utilized a backcrossing breeding approach with commercial "Kyokai" yeasts and natural yeast isolates.
• Performed three rounds of crossing procedures to generate homozygous fdc1 crossbreed strains.
• Assessed 4-VG levels in the culture supernatant of the resulting hybrid strains compared to parental strains.

Main Results:

• Successfully generated yeast strains homozygous for non-functional fdc1 alleles.
• Confirmed a significant reduction in 4-VG levels in the supernatant of the homozygous fdc1 hybrid strains.
• The breeding approach did not rely on growth selection for the desired fdc1 mutation.

Conclusions:

• A precise breeding method was established to remove specific gene alleles, like wild-type FDC1, from yeast.
• This approach effectively reduces 4-VG formation in sake by utilizing homozygous fdc1 yeast strains.
• The technique holds potential for designing custom yeast strains for various applications by leveraging historical yeast diversity.